Polyhydroxylated compounds, such as oligosaccharides, complex carbohydrates and lipid and protein conjugates thereof, are molecules of great importance in biochemical processes of biological recognition such as cell adhesion, viral infections, cell differentiation in organ development and metastasis (Koeller, K. M., Wong, C. H., Nat. Biotechnol. 18 (2000) 835). Thus, the enzymes involved in their synthesis or degradation, glycosyltransferases and glycosidases respectively, constitute inhibition or activation targets (according to Kolter, T., Wendeler, M., Chembiochem 4 (2003) 260) since they are involved in metabolic disorders and diseases, such as type II diabetes, hepatitis B and C, Gaucher's disease, Fabry's disease, cystic fibrosis, colon cancer, or viral infections including HIV (Asano, N., J. Enzyme Inhib. 15 (2000) 215; Asano, N., Glycobiology 13 (2003) 93R; Fiaux, H., Popowycz, F., Favre, S., Schutz, C., Vogel, P., Gerber-Lemaire, S., Juillerat-Jeanneret, L., J. Med. Chem. 48 (2005) 4237).
Among the polyhydroxylated compounds which are inhibitors of glycosyltransferases and glycosidases, the types of iminocyclitols which stand out are pyrrolidines, piperidines, indolizidines, pyrrolizidines, nortropanes, and seven-membered polyhydroxylated iminocyclitols), among others, some of them being powerful inhibitors of glycosidases and glycosyltransferases. (Asano, N., J. Enzyme Inhib. 15 (2000) 215; Asano, N., Glycobiology 13 (2003) 93R; Lillelunh, V. H., Jensen, H. H., Liang, X., Bols, M., Chem. Rev. 102 (2002) 515; Compain, P., Martin, O. R., Curr. Top. Med. Chem. 3 (2003) 541; Mehta, G., Lakshminath, S., Tetrahedron Lett. 43 (2002) 331; Moris-Varas, F., Qian, X. -H., Wong, C. -H., J. Am. Chem. Soc. 118 10 (1996) 7647; Fuentes, J., Olano, D., Pradera, M. A., Tetrahedron Lett. 40 (1999) 4063; Li, H. Q., Bleriot, Y., Chantereau, C., Mallet, J. M., Sollogoub, M., Zhang, Y. M., Rodriguez-Garcia, E., Vogel, P., Jimenez-Barbero, J., Sinay, P., Org. Biomol. Chem. 2 (2004) 1492; Lin, C. C., Pan, Y. S., Patkar, L. N., Lin, H. M., Tzou, D. L. M., Subramanian, T., Bioorg. Med. Chem. 12 (2004) 3259; Godin, G., Garnier, E., Compain, P., Martin, O. R., Ikeda, K., Asano, N., Tetrahedron Lett. 45 (2004) 579).
Some derivatives such as miglitol and miglustat are drugs marketed for the treatment of type II diabetes (Platt, F. M., Butters, T. D., Drugs 63 (2003) 2435).
Some natural iminociclytols or plant extracts containing them have also been described as functional ingredients in the food industry or as dietary supplements. Thus, US20010018090 discloses the use of 1-deoxynojirimicin or an analogue thereof as a calorie reducing agent that may be incorporated in food or beverage; US20060222720 discloses an anorectic agent containing aqueous solvent extracts of Vernonia cinerea and mulberry as active ingredients; WO2004037001 discloses the addition of mulberry extracts to a sacharide containing food for regulating blood sugar levels.
So far, the chemoenzymatic strategies for the synthesis of iminocyclitols disclosed are based on the use of aldolases, enzymes capable of catalyzing stereoselective aldol condensation reactions between aldehydes and ketones. (Von der Osten, C. H., Sinskey, A. J., Barbas, C. F., III, Pederson, R. L., Wang, Y. F., Wong, C. H., J. Am. Chem. Soc. 111 (1989) 3924, Romero, A., Wong, C. H., J. Org. Chem. 65 (2000) 8264, Look, G. C., Fotsch, C. H., Wong, C. H., Acc. Chem. Res. 26 (1993) 182, Machajewskif, T. D., Wong, C. H., Angew. Chem. Int. Ed. 39 (2000) 1353; patent (US005329052A)). Among known aldolases, the dihydroxyacetonephosphate (DHAP)-dependent aldolases have focused the attention due to four reasons:                1) their availability, either because some of them are commercially available or because their preparation is relatively easy from modified E. coli,         2) their high stereoselectivity,        3) their wide structural tolerance for the acceptor aldehyde, and        4) their stereogenic ability.        
DHAP-dependent aldolases (DHAP-aldolases) catalyze reversible DHAP aldol addition with an acceptor aldehyde, obtaining α,β-dihydroxyketones with two new stereogenic centres. It is especially interesting to note that the four stereocomplementary DHAP-aldolases (FIG. 1) are already known: D-fructose-1,6-diphosphate aldolase (FruA); L-rhamnulose-1-phosphate aldolase (RhuA); L-fuculose-1-phosphate aldolase (FucA); and D-tagatose-1,6-diphosphate aldolase (TagA). Advantageously, these biocatalysts have some ability to control the aldol addition stereochemistry, the configuration of the new generated stereogenic centres depending on the enzyme and not on the reagents.
The general chemoenzymatic synthetic scheme of iminocyclitols synthesis using DHAP-aldolases is shown in FIG. 2. The critical step of this scheme is the aldol addition of DHAP to aminoaldehydes or synthetic equivalents thereof catalyzed by DHAP-aldolases. In this step two stereogenic centres, whose configuration depends on the enzyme, are generated, although there are numerous examples wherein, depending on the substrate, the enzyme looses selectivity, obtaining diastereomeric products. The following step is a hydrolysis of the phosphate moiety of the aldol adduct by an acid phosphatase. Finally, the Cbz removal and the transformation to iminocyclitol is generally carried out in one step.
The preparation of the dihydroxyacetonephosphate (DHAP) is a critical step of this synthesis. The chemical synthesis of dihydroxyacetonephosphate is carried out through five steps with overall yields about 60% (FIG. 3) according to Jung et al. disclosure (Jung, S. -H., Jeong, J. -H., Miller, P., Wong, C.-H., J. Org. Chem. 59 (1994) 7182).
Multienzyme systems for “in situ” generation of DHAP are an alternative approach. These are sophisticated processes demanding a very fine control of the reaction conditions and the presence of components in the reaction mixture which can hinder the isolation and purification of the final product (Fessner, W. D., Sinerius, G., Angew. Chem. Int. Ed. 33 (1994) 209; Charmantray, F., El Blidi, L., Gefflaut, T., Hecquet, L., Bolte, J., Lemaire, M., J. Org. Chem. 69 (2004) 9310, Sanchez-Moreno, I., Francisco Garcia-Garcia, J., Bastida, A., Garcia Junceda, E., Chem. Commun. (2004) 1634).
In the patent (US005329052A) and as it is disclosed by Von der Osten et al. (Von der Osten, C. H., Sinskey, A. J., Barbas, C. F., III, Pederson, R. L., Wang, Y. F., Wong, C. H., J. Am. Chem. Soc. 111 (1989) 3924), dihydroxyacetone is used in the presence of arsenic salts as a substitute of DHAP for enzymatic aldol addition. Although the process is simplified, the use of arsenic salts is not applicable due to their toxicity and, therefore, their environmental and health danger.
Already disclosed chemoenzymatic synthesis of iminocyclitols use about 8 steps from the acceptor aldehyde: two of them are enzymatic steps, the aldol addition of DHAP to the aldehyde, and the phosphate ester hydrolysis; and 6 chemical steps for DHAP synthesis, and the formation of the corresponding iminocyclitols. If the reaction is carried out with multienzyme systems, it requires two more enzymes: one for the formation of the key intermediate, DHAP, and another for regenerating the enzymatic phosphorilation reagent. Therefore, these are strategies with a lot of steps or very sophisticated and therefore with a limited industrial applicability.